The major neurological consequence of cocaine abuse is inhibition of dopamine reuptake. As a consequence, cocaine abuse has been found to disrupt plasma prolactin levels. Availability of a cell culture system for studying dopaminergic repression of the prolactin gene would help understand how cocaine disrupts physiological regulation of this gene. However, since GH rat pituitary cell lines, previously employed for many studies of prolactin gene regulation, lack dopamine receptors (D2R), little is yet known about how dopamine represses expression of the prolactin gene. We have recently shown that expression in GH3 cells of either of two forms of the D2R receptor, D2L or D2S, permits dopaminergic regulation of a co- transfected rat prolactin-CAT construct. The major aim of the proposed research is to use this system to elucidate the cellular pathway that mediates dopaminergic repression of the prolactin gene. We plan first to investigate whether a dopaminergic agonist, quinpirole, can regulate prolactin gene transcription in a GH3 cell line stably expressing transfected D2L cDNA. We will then employ promoter-CAT constructs containing fragments of the prolactin promoter, transiently transfected into this cell line, to localize and characterize a dopaminergic repressor element (DARE) in the prolactin promoter. Further investigation at the DNA level will depend upon whether these initial studies implicate the pituitary-specific transcription factor pit-1 in dopamine action. If so, dopamine-induced modifications of pit-1 will be investigated; if not, the gene-proximal dopamine mediator will be cloned. More gene-distal mediators (both small molecules and macromolecules) of dopaminergic repression will be investigated by employing: (1) transfection as above to determine whether a DARE co-localizes with a regulatory element for Ca2+ and/or cyclic AMP; (2) pharmacological studies of dopaminergic regulation of endogenous prolactin gene expression. Finally, we will use similar approaches to identify and characterize a DARE in the mouse fos gene promoter.